SHISA7 (CKAMP59) has emerged as one of the most intriguing new members of the SHISA family, in that, unlike other CKAMP counterparts, it exhibits a direct function in inhibitory synaptic GABAAR regulation. We used bioinformatics and experimental methods in this research to explore competing endogenous RNA (ceRNA) regulation of BCAS4 and SHISA7 in tau pathogenesis and their capacity as peripheral biomarkers linked to an abnormal inflammatory response in AD. The Gene Expression Omnibus database included two microarray datasets, including information on mRNAs (GSE106241) and miRNAs (GSE157239) from individuals with AD with different degrees of AD-associated neurofibrillary pathology in the temporal cortex (TC) tissue specimens and corresponding controls were downloaded from the Gene Expression Omnibus database. The limma package in the R software was used to identify differently expressed mRNAs (DEmRNAs) and miRNAs (DEmiRNAs) associated with AD-related neurofibrillary pathology. Additionally, we used the quantitative polymerase chain reaction technique to examine the expression of the BCAS4/hsa-miR-185-5p/SHISA7 ceRNA axis in the peripheral blood (PB) of fifty AD patients and fifty control subjects. BCAS4 was shown to act as a ceRNA to control the SHISA7 expression throughout AD-associated neurofibrillary pathology in TC tissue specimens by sponging hsa-miR-185-5p, based on our bioinformatics study. Furthermore, in PB specimens from individuals suffering from AD and normal controls, we found no substantial differences in BCAS4 expression patterns. SHISA7 expression in AD patients’ PB was found to be reduced, as was the case in the TC. On the other hand, we discovered reduced amounts of hsa-miR-185-5p in AD patients’ PB samples compared to control subjects, unlike in TC tissue, where it had been demonstrated to be overexpressed. BCAS4 and SHISA7 expression levels showed a strong positive correlation, suggesting the presence of an interconnected network, most likely as a result of ceRNA regulation among PB specimens. The present study is the first evidence to highlight the expression of the BCAS4/miR-185-5p/SHISA7 ceRNA axis in the brain and PB of AD patients, and offers a new viewpoint on molecular processes underlying AD pathogenic mechanisms.Alzheimer’s disease (AD) is a heterogeneous degenerative brain disorder with a rising prevalence worldwide.  MAPT) gene encodes tau, a microtubule-associated protein primarily generated in neurons. Intracellular tau is occasionally hyperphosphorylated, forming dangerous oligomers and observable aggregates as NFTs (Alzheimer’s disease (AD) is known as a type of dementia and a progressive neurodegenerative disorder (NDD), causing memory, thinking, and behavioral problems . Accordi as NFTs . During  as NFTs . The pat as NFTs . Aβ and  as NFTs . Moreove as NFTs . Further as NFTs . As a re as NFTs . Irrespe as NFTs .SHISA7 (CKAMP59) has emerged as an intriguing SHISA family because, unlike other CKAMPs, Shisa7 directly affects the regulation of GABAARs at inhibitory synapses . A chip-based platform GPL24170 Agilent-044312 Human 8 × 60K Custom Exon array (Probe Name version) was used for the dataset. The GSE106241 included 60 human TC tissue samples split into seven groups according to Braak staging  reflecting the severity of the disease  tissue samples with different degrees of AD-related neurofibrillary pathology (GSE106241). We intended to identify expression changes of  disease . Braak s disease .2  < 0.01, and (BCAS4 and SHISA7 genes were created using the Enhanced Volcano (version 1.8.0) and Pheatmap (version 1.0.12) R packages.Using the normexp method, background correction was performed  with an 2 . Studentfollows:  a false .01, and  | log2 ft-test was used to identify statistically significant miRNAs. Also, the cut-off for aberrantly expressed miRNAs were set as follows:  < 0.01, and  linked to AD-related neurofibrillary pathology, we employed a bioinformatics method identical to the one described above. GSE157239 miRNA profile data were acquired from the NCBI GEO. The platform GPL21572 (miRNA-4) Affymetrix Multispecies miRNA-4 Array (ProbeSet ID version) was applied for the dataset. The GSE157239 comprised eight TC samples from AD patients (Braak stage III or above) and eight from control subjects. The Robust Multichip Average (RMA), an effective tool in the affy Bioconductor package, was used for background correction and quantile normalization of the entire raw data files . An intefollows:  a false .01, and  | log2 fBCAS4/SHISA7  to identify interactions among miRNAs linked to AD-related neurofibrillary pathology with 4/SHISA7 . BindingBCAS4 and SHISA7 in the ceRNA regulatory axes. The Hmisc and psych packages were used to calculate the correlations and visualization. The ceRNA regulatory axes were built according to the co-expression relation and miRNA-mRNA interactions.The Pearson correlation analysis was performed to determine if there were any positive correlations between This case-control research included 100 individuals, 50 AD patients as well as 50 healthy controls who were gender and age-matched. The present investigation was approved by Tabriz University of Medical Sciences’ clinical research ethics committee . The participants were recruited from the Department of Neurology of Tabriz University of Medical Sciences’ Imam Reza Hospital. A neurology specialist identified the individuals using the Diagnostic and Statistical Manual of the American Psychiatric Association (DSM-V) criteria . The criU6 and Ubiquitin C (UBC) were employed as internal controls to normalize miRNA and mRNA levels, respectively. The Step OnePlus™ Real-Time PCR and the RealQ Plus2x Master Mix  were used for the qPCR. All qPCR reactions were performed in duplicate.Total RNA was isolated from whole blood utilizing the Hybrid-R™ Blood RNA purification kit according to the manufacturer’s instructions  and treated with DNase I to remove DNA contamination. NanoDrop was employed to assess the quantity and quality of extracted RNA . The synthesis of cDNA was done by the cDNA synthesis Kit (GeneALL) according to the manufacturer’s guidelines. The cDNA was stored at −20°C for further investigation. The primer sequences used in reverse transcription, as well as qPCR reactions, are listed in BCAS4, SHISA7, and hsa-miR-185-5p in AD patients and healthy controls, as well as subgroups. Age and gender impacts were adjusted. The adjusted P-values of less than 0.05 were considered significant. The expression of the abovementioned genes was also studied across age groups and between males and females. The Spearman correlation coefficients were utilized to evaluate the connections between the variables in the research. The genes’ diagnostic power was determined using a receiver operating characteristic (ROC) curve analysis.The data analysis was performed using the R v.4 software packages brms, stan, pROC, and GGally. The Bayesian regression model was employed to investigate the relative expressions of Background correction, batch modification, gene filtering, and normalization were all performed prior to DEGA. The AgiMicroRna Bioconductor package was utilized to monitor the quality. The gene expression dataset’s box plots were used to evaluate the distribution of data after normalization . DistincBCAS4 was significantly downregulated in Braak stages I-VI, while SHISA7 was significantly downregulated only in Braak stage V. Thus, Braak stage V was the only one in which both the BCAS4  and the SHISA7  genes were dysregulated. P-value < 0.01, and  and 14 miRNAs  obtained from GSE157239 dataset reanalysis may target BCAS4 and SHISA7, respectively. Of those miRNAs, hsa-miR-185-5p targeted both genes BCAS4 and SHISA7. The hsa-miR-185-5p expression levels in TC samples of AD were statistically higher than those in controls .We employed miRWalk database to find interactions between miRNAs linked to AD-related neurofibrillary pathology and BCAS4 and SHISA7 to validate the ceRNA axes theory, which states that ceRNAs are positively regulated by each other via interactions with miRNAs. A significant positive correlation was found between the levels of expression of the evaluated genes   . Based oFollowing consideration of the inclusion and exclusion criteria, 50 AD patients  with age [mean ± standard deviation (SD)] of 76.36 ± 6.26 and 50 healthy controls  with age (mean ± SD) of 74.3 ± 6.22 were included in the study. The MMSE scores (mean ± SD) of the patient and control groups were 14.2 ± 5.94 and 27.5 ± 0.76, respectively.BCAS4, SHISA7, and hsa-miR-185-5p genes’ relative expression levels in patients with AD and controls. The BCAS4 expression levels demonstrated no significant differences in PB samples among AD patients and healthy controls , as well as subgroups. SHISA7 expression was significantly lower in PB samples from AD patients compared to controls . Such decreased expression was found between male and female subgroups too . The hsa-miR-185-5p expression was found to be significantly lower  when PB samples from AD patients were compared to controls. Male and female subgroups also had lower expression . BCAS4, SHISA7, and hsa-miR-185-5p, respectively.BCAS4, SHISA7, and hsa-miR-185-5p expressions were not correlated to the patients’ age. In AD patients, expression of BCAS4 were correlated positively with SHISA7 . In the PB of patients, our correlation results between hsa-miR-185-5p and SHISA7 showed a weak positive correlation  instead of a negative correlation. Furthermore, the expressed levels of hsa-miR-185-5p and BCAS4 were not correlated significantly in AD cases  (The diagnostic power of ve (AUC) .BCAS4/SHISA7 verified ceRNA axis in AD development.According to growing research-based data, GABAergic neurotransmission faced serious pathological alterations in AD, and it might be a successful therapeutic candidate for this NDD . MoreoveBCAS4 could serve as a ceRNA to regulate the expression of SHISA7 in AD-related neurofibrillary pathology via sponging hsa-miR-185-5p. To our knowledge, this would be the first report of a possible role of the BCAS4/hsa-miR-185-5p/SHISA7 axis in the tau pathology of AD.Our bioinformatics analysis showed that in silico analysis showed that the expression levels of BCAS4 were substantially lower in TC samples from AD patients and the control group. BCAS4 is a new gene, which is cloned from breast cancer cells, encoding a cytoplasmic protein (211 aa) with no substantial homology to known proteins . They hypothesized that the development of this novel 6 mer MRE would lead to the observed GRN downregulation in LOAD  besides feed-forward regulation provoked by widely known transcription factors (resulting in positive miRNA-mRNA correlations)   functions in the SHISA7 3′ UTR (hsa-miR-185-5p functions as an enhancer in AD patients’ PB.Expression levels of lations) . The hsa murine) . This fi7 3′ UTR . More stSHISA7 and hsa-miR-185-5p had a diagnostic value of 0.758 and 0.779, in turn, in differentiating patients with AD from healthy subjects. Because of the small sample size, the obtained findings should be interpreted with caution. If future research supports the findings of the present study, the SHISA7 and hsa-miR-185-5p transcription levels might be utilized as AD-associated markers.Eventually, we determined that SHISA7, BCAS4, and hsa-miR-185-5p in the PB cell subpopulation.Our research has several limitations. Firstly, various aspects, such as diverse methods, sample preparation, platforms, data analysis, and patient characteristics, may impact expression patterns of genes. Second, a limited sample size might lead to a lack of statistical validity. Furthermore, our bioinformatics analysis must be verified by confirmatory experimental techniques. Finally, we did not examine expressions of BCAS4/miR-185-5p/SHISA7 ceRNA axis in the brain and PB of AD patients. The obtained results are preliminary, and further in vitro and in vivo research might strengthen these results. Whereas the possible roles of this ceRNA axis require more exploration, this research advances the current insights into the GABAergic system associated with GABAAR and provides a novel viewpoint on the molecular processes behind AD development.Competing endogenous RNA regulation has biologically significant consequences in a variety of illnesses, which can help to explain pathogenic processes and provide possibilities for novel treatments. As a result, our attempts to comprehend various aspects of ceRNA regulation processes in AD pathogenesis give new insights into possible molecular targets and lead to the discovery of ceRNA-based biomarkers. The present study is the first evidence to highlight the expression of the The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Tabriz University of Medical Sciences’ Clinical Research Ethics Committee . The patients/participants provided their written informed consent to participate in this study.HS, MR, and MoT wrote the draft and revised it. SA-J and JG analyzed the data. MA, AJ, and MaT performed the experiments. BH and RJ collected the data and corresponding clinical information. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lombardy was the most affected Italian region by the first phase of the COVID-19 pandemic and underwent urgent reorganization for the management of emergencies, including subarachnoid hemorrhage from a ruptured cerebral aneurysm (aSAH). The aim of the study was to define demographics, clinical, and therapeutic features of aSAH during the COVID-19 outbreak and compare these with a historical cohort.In this observational multicenter cohort study, patients aged 18 years or older, who were diagnosed with aSAH at the participating centers in Lombardy from March 9 to May 10, 2020, were included (COVID-19 group). In order to minimize bias related to possible SAH seasonality, the control group was composed of patients diagnosed with aSAH from March 9 to May 10 of the three previous years, 2017–2018-2019 (pre-pandemic group). Twenty-three demographic, clinical, and therapeutic features were collected. Statistical analysis was performed.p-value = 0.030), while “therapeutic delay” did not differ significantly between the two periods . Patients with poor outcome (GOS at discharge from 1 to 3) were higher during the COVID-19 period (54.2%) compared to pre-pandemic . In logistic regression analysis, in which outcome was the dichotomized Glasgow Outcome Scale (GOS), five variables showed p-values < 0.05: age at admission, WFNS grade, treatment (none), days in ICU, and ischemia.Seventy-two patients during the COVID-19 period and 179 in the control group were enrolled at 14 centers. Only 4 patients were positive for SARS-CoV-2. The “diagnostic delay” was significantly increased (+ 68%) in the COVID-19 group vs. pre-pandemic (1.06 vs. 0.63 days, respectively, We documented a significantly increased “diagnostic delay” for subarachnoid hemorrhages during the first COVID-19 outbreak in Lombardy. However, despite the dramatic situation that the healthcare system was experiencing, the Lombardy regional reorganization model, which allowed centralization of neurosurgical emergencies such as SAHs, avoided a “therapeutic delay” and led to results overall comparable to the control period. Subarachnoid hemorrhage from a ruptured cerebral aneurysm (aSAH) is a time-dependent disease and a neurosurgical emergency. Some authors have reported an overall decrease in the number of cases admitted for aSAH during the first phase of the SARS-CoV-2 pandemic , 6, 12, During the first phase of the COVID-19 pandemic, the Lombardy region, which is the most populated region in Italy with nearly 10 million inhabitants, was the most affected. The Lombard Regional Council organized an emergency network during the national lockdown, creating an unprecedented Hub and Spoke system: treatment of time-dependent emergency was centralized in Hub hospitals and COVID-19 patients were treated in different satellite reinforced Spoke hospitals. All deferrable elective activities were suspended. As far as neurosurgery is concerned, three Hub centers were identified, each covering a “macro-zone” with a catchment area of more than 3 million people: Milan and hinterland , north and central Lombardy , and south and east Lombardy  , 39. MosAs defining the epidemiological characteristics, management, treatment, and outcomes of aSAH in Lombardy during the COVID-19 outbreak has important scientific relevance and possible practical implications, this observational multicenter study was carried out. The main endpoint of the study was evaluation of whether the COVID-19 pandemic influenced management and outcomes of aSAH compared to the pre-pandemic period. Secondary endpoints included delay in diagnosis or treatment and differences in type of treatment between the two periods.This was an observational multicenter cohort study with a control group in a 1:3 ratio. Patients aged 18 years or older, who were diagnosed with aSAH at the participating centers in Lombardy from March 9 to May 10, 2020 , were included (COVID-19 group). All patients underwent screening for SARS-CoV-2 with reverse transcriptase–polymerase chain reaction assay in a nasopharyngeal swab. Chest radiography and/or chest computed tomography (CT) scan were performed to investigate any pulmonary abnormalities.In order to minimize the bias related to possible SAH seasonality, the control group was composed of patients diagnosed with aSAH from March 9 to May 10 of the three previous years, 2017–2018-2019 ( pre-pandemic group).The following data were recorded for all patients Table : age, seThe study was approved by the local ethics committee (NP 4192 — SAH-COVID-LOMB). Patient consent was obtained at the time of treatment.P-values of the two proportions z-test are reported for subgroups of patients defined by the following variables: WFNS grade, Fisher grade, aneurysm location, treatment, and GOS discharge. In case of quantitative variables, the Mann–Whitney test, which identifies if two independent subsamples come from the same population, was used  without any significant difference (p = 0.122). Only 4 patients were positive for SARS-CoV-2 (5.5% of subjects in COVID-19 period); hence, this variable was not considered in data analyses.Seventy-two patients during the COVID-19 period [45 female (62.5%) vs. 27 male (37.5%)] and 179 in the control group [122 female (68.2%) vs. 57 male (31.8%)] were enrolled at 14 centers. There was no relevant differences in gender between the two groups . The “diagnostic delay” was significantly increased (+ 68%) during the COVID-19 group . When the “diagnostic delay” was dichotomized as “same day” vs. “at least one day,” 24 patients (33.3%) were diagnosed after at least 1 day during the COVID-19 pandemic compared to 34 patients (20.7%) in the pre-pandemic period (p = 0.051). “Therapeutic delay” in days did not differ significantly between the COVID-19 and pre-pandemic periods .In bivariate analysis, which compared the COVID-19 vs. pre-pandemic periods (Table p = 0.044). Particularly, patients with GOS 2 were 12.5% in COVID-19 vs. 4.5% in pre-pandemic period (p = 0.022), while patients with GOS 5 were 27.8 in COVID-19 compared to 47.5% in pre-pandemic period (p = 0.004). No significant differences were seen in the type of treatment  between the two groups (p = 0.701).The percentage of patients with poor outcome (GOS at discharge from 1 to 3) was higher during the COVID-19 period (54.2%) compared to pre-pandemic , the collinearity problem was excluded obtaining reliable and stable estimates of regression coefficients.Before proceeding, the Spearman correlation coefficient between quantitative variables in the model (age at admission and days in ICU) was computed. Since it equals to 0.012  was five times higher in patients with ischemia compared to those without it . In the multivariate logistic model, period (COVID-19 vs. pre-pandemic) was not associated with dichotomized GOS at discharge . Additionally, the bivariate analysis, which assessed the association between GOS at discharge and period (COVID-19 vs. pre-pandemic), was not statistically significant if the four patients who tested positive for SARS-CoV-2 were excluded from the COVID-19 period (p = 0.072).The results of logistic regression analysis are reported in Table This observational multicenter cohort study enrolled 251 patients diagnosed with aSAH in two reference periods. The two subgroups, COVID-19 and pre-pandemic, were quite homogeneous: there were no significant differences in age, gender, previous comorbidities , WFNS and Fisher at admission, and aneurysm location (Table p = 0.030). Both hospital overcrowding [p = 0.343).From our data, the first relevant issue is a statistically significant diagnostic delay  in the COVID-19 period : there was not a “therapeutic delay” (time between diagnosis and treatment of the aneurysm) during the organization in Hub and Spoke in the COVID-19 period. It can therefore be inferred that, despite the difficulties for patients to reach healthcare facilities or for the healthcare system to manage the pre-hospital emergency, once a diagnosis was obtained, each patient received timely treatment, just as before the pandemic, even if they were transferred from Spoke centers to Hub centers. It is difficult to say what could have happened with a different organization, but considering the amount of resources required by the pandemic, with the conversion of many neurosurgeries into COVID wards, a number of emergencies, including vascular ones, probably could not have been adequately addressed.No differences were recorded in the time to treatment in the two periods . This might be explained by either the need of reducing the ICU time as a sign of pressure on the hospital system or as a sign of optimization in high volume centers. The study was not designed to investigate this aspect. As expected, patients who did not undergo treatment, mainly because of poor prognosis at admission, had a significantly worse outcome compared to treated patients.ICU and total hospitalization days were significantly less in the COVID-19 period ; this small difference was, however, no longer evident after excluding the 4 patients who were positive for SARS-CoV-2 (p = 0.072) and it was not significant in the multivariate logistic model (Table p = 0.052). From this point of view, it could be argued that the new organization into a Hub and Spoke system worked adequately: the COVID-19 period itself did not influence outcome of patients with aSAH. This study further confirms that age and poor WFNS at admission, together with ischemia, are negative prognostic factors.In the multivariate logistic model, age at admission, WFNS grade, days in ICU, and ischemia were prognostic factors for poor outcome GOS 1–3). The COVID-19 period was slightly significant in the descriptive statistics stratified for GOS (Table . The COVAs far as SARS-CoV-2 infection itself is concerned, we could not determine the effect it may have had on SAH outcomes, since during the COVID-19 period, only 4 patients tested positive for SARS-CoV-2. We can, however, observe that all these patients but one had a GOS between 1 and 2. Higher mortality in patients with SAH and COVID-19 compared to those without COVID-19 has already been reported, due to a higher rate of systemic comorbidities such as pulmonary embolism, acute coronary syndrome, and respiratory failure , 31. TheGiven the high early lethality of aSAH and the statistically significant diagnostic delay that was documented during the COVID-19 period, it is not possible to exclude that some patients died before hospital admission; the study was designed only to investigate hospital admission of aSAH.This study documented a significantly increased diagnostic delay for aSAH during the first COVID-19 outbreak in Lombardy, possibly due to patients’ fear of hospitalization. Despite the dramatic situation that the healthcare system was experiencing, the Hub and Spoke organization model, with centralization of neurosurgical emergencies, was not associated with therapeutic delay and, even in the presence of clear signs of system overload, led to results overall comparable to the control period in the management of aSAH.


As a result, HIIT-R and HIIT-F stimulated similar improvements in VO2max . Only the HIIT-F group revealed significant improvements in muscle performance . Body fat percentage decreased  in both HIIT-R and HIIT-F with no between-group differences. We concluded that HIFT was equally effective in promoting body composition and aerobic fitness compared to HIIT-R. HIFT resulted in improved muscle performance, whereas the HIIT-R protocol demonstrated no gains.High-intensity interval running (HIIT-R) and high-intensity functional training (HIFT) are two forms of HIIT exercise that are commonly used. The purpose of this study was to determine the effects of HIFT on aerobic capacity and body composition when compared to HIIT-R in females. Twenty healthy, untrained female university students (age 20.5 ± 0.7 year) were randomly assigned to a 12-week HIIT-R or HIFT intervention. The HIIT-R group involved a 30 s maximal shuttle run with a 30 s recovery period, whereas the HIFT involved multiple functional exercises with a 2:1 work-active recovery ratio. Body composition, VO Regular physical activity (PA) is beneficial for health ,2,3. DesHIIT involves repeated bouts of high-intensity exercises separated by a recovery using low-intensity activities or inactivity . Recent Originally, HIIT was used to improve the performance of endurance athletes . CyclingThe intrinsic factors of participants are also important when considering exercise adherence ,19. SeveHigh-intensity functional training (HIFT) has become a relatively popular training modality in recent years and is an alternative to traditional aerobic activities. The HIFT protocol consists of a variety of functional movements that are executed at a high intensity ,23). RecAlthough studies have shown that HIFT has similar or superior benefits for physical fitness compared to moderate-intensity continuous training and have indicated more enjoyment compared to HIIT, the question remains as to whether HIFT is as efficient as HIIT for improving health-related fitness.2max, and muscle endurance following a 12-week intervention compared to changes achieved using a running-based HIIT. The purpose of this study was to investigate the effects of different kinds of training on fitness parameters in untrained female university students. It was hypothesized that (a) aerobic fitness would be increased in both the HIIT-F and HIIT-R groups; (b) that fat mass would be decreased in both the HIIT-F and HIIT-R groups; and (c) that muscular strength and endurance would be improved in the HIIT-F group.While HIFT is not synonymous with HIIT, they share an important conceptual commonality in the modality of both being of a high intensity. The current study was undertaken to clarify how a functional exercise based on HIIT would improve fitness parameters such as fat mass, blood pressure, VOn = 10) or a functional training-based HIIT (HIIT-F) (n = 10). The participants were nonsmokers and were instructed to maintain their normal dietary intake and lifestyle habits  throughout the intervention. Nutritional supplements and intense exercise beyond their usual exercise habits were forbidden during the intervention period [Twenty untrained healthy females who were physical inactive volunteered to participate the study. Participants who did not exercise for more than 2 h weekly for at least 12 months were considered as physically inactive . All of n period . All of A randomized controlled trial was used in this study. Each participant completed twelve weeks of 36 sessions of HIIT-R or HIIT-F intervention (three sessions per week) comprising a total of 19 min per session . All sessions were conducted and monitored at the same indoor stadium and at the same time of day between 9:00–10:00 a.m. Heart rates (HR) were collected with an activity wristband  during each session to ensure that the required high intensity was achieved. The reliability and validity of the heart rate index and distance index were reported in a previous study . The actParticipants were instructed to arrive at the laboratory 9:00 a.m. after a normal breakfast. Before the measurements were taken, participants were asked to empty their bladder to minimize measurement errors caused by “electrically silent” . Under t2max [2max, for untrained participants, submaximal exercises can be used as a reliable measure to estimate this value. Cooper’s 12 min running test was used to assess aerobic fitness in this study. All of the participants completed two trials of the running test separated by 24 h of rest. After a 5 min warm up, the participants were required to wear an activity wristband  and commenced running on a standard 400-metre running track. Subjects were instructed to run as many laps as possible on a standard outdoor track during the 12 min test period. All of the participants were encouraged verbally and were instructed to focus on their own pace throughout the test. The experimenter verbally provided the elapsed time at 3, 6, and 9 min. At the end of the 12 min period, the experimenter called “stop”. All of the participants ceased running and stood still, until the distance achieved, and maximal heart rate (HRmax) were recorded. The HRmax displayed on the activity wristband was recorded immediately upon the cessation of exercise, and the higher value of the two trials was used for analysis. The total distance run was determined by measures obtained from the activity band. An estimated VO2max was calculated using Cooper’s standardized equation [2max was highly correlated with the laboratory-determined one and had acceptable reliability and validity (r = 0.897) [2max.The most reliable and effective way to measure aerobic capacity is to record each individual subject’s VO2max . Althougequation . The cal= 0.897) . The aveMuscle performance was assessed using a field-based muscle fitness test battery. Timed sit-ups, push-ups with flexed knee , and standing broad jump were recommended by previous studies to assess muscle performance ,39,40,41Exercise interventions commenced one week after the last measurement day. Both the HIIT-R and HIIT-F interventions were conducted three days per week on Mondays, Wednesdays, and Saturdays for twelve weeks. If the participants were unable to attend a scheduled exercise day, the exercise was performed on the next day and was monitored by the same researcher.Participants in the HIIT-R group were required to complete 144 repetitions of maximal shuttle running for a total exercise time of 72 min. Each bout included a 30 s maximal shuttle run between cones placed 20 m apart with a 30 s recovery period between runs. The validity and reliability of 40 m maximal shuttle run as a measure of anaerobic performance has been reported previously . The parParticipants in the HIIT-F group performed multiple functional exercises using their own body weight based on Tabata training . AccordiThe training frequency was the same as the HIIT-R group. All training exercises were recorded by video, which was provided to the HIIT-F participants prior to intervention to ensure that they were familiar with the movements and procedures. This video was played on a screen during the training intervention to ensure that the participants kept up with the rhythm of each movement.To ensure that the interventions were performed at adequate exercise intensity, participants’ HRs were recorded throughout the session with an activity wristband. The peak heart rate (HR peak) of each session was considered to be 75% or more of the HRmax that had been recorded during Cooper’s 12 min running test. All of the sessions began with a standardized 10 min low-to-moderate running and stretching followed by maximal shuttle run or functional training and ended with a 5 min cool-down and stretching.p < 0.05.Statistical analyses were performed using SPSS, version 23.0 . Data were presented as means x± SD. A two-factor analysis of variance with repeated measures was used to analyze differences in body composition, muscle performance, and aerobic capacity, with intervention (pretraining and post training) as a within-group factor and group (HIIT-R and HIIT-F) as a between-group factor. A significant intervention x group interaction was used to identify training-induced changes in body composition, muscle performance and aerobic capacity. Data were subsequently checked by Tukey’s post hoc test if a significant interaction was revealed. Furthermore, paired t-tests were used to estimate within-group effects, and independent t-tests were conducted to examine differences between groups. The significance level was established as All of the participants completed all of the sessions over the twelve-week period. There were no significant between-group differences in the variables measured at baseline .Body Compositionp < 0.05) in the percent body fat for both groups (p > 0.05). Body mass index (BMI) (p > 0.05). Lean muscle mass increased in both groups .Body composition data are presented in h groups a, with nex (BMI) b and waiex (BMI) c did notResting Heart Rate and Blood Pressurep < 0.05) was improved compared to baseline in both intervention groups, while no interaction effect was observed. Resting systolic BP and diastolic BP remained unchanged (p > 0.05) after training in both the HIIT-R and HIIT-F groups.Resting HR  = (distance(m)-506)/45. VO2max data for all participants are presented in p < 0.05) in the VO2max was demonstrated in both training groups compared to baseline measures, while no significant intervention x group interaction was revealed between HIIT-R and HIIT-F after intervention compared to baseline (p > 0.05). The extent of the change in VO2max was 17.1% ± 5.6% and 12.7% ± 6.7% in the HIIT-R and HIIT-F groups, respectively.VObaseline c. (p > 0Muscle Performancep < 0.05) by 16.5% ± 3.1% and the distance obtained in the stand broad jumping test improved (p < 0.05) by 5.1% ± 2.2%, whereas these variables were unaltered (p > 0.05) in the HIIT-R group. Flexed push-ups were unaltered in both the HIIT-R and HIIT-F groups  without significant changes in the body mass (p = 0.064 for HIIT-R and p = 0.051 for HIIT-F). The non-significant change in BMI may be due to the following reasons: the insufficient exercise duration per session (2 min vs. 6–10 min); the uncontrolled dietary intake during the intervention; and the characteristics of the participants regarding body weight. This suggestion has been highlighted in a recent systematic review [However, current research has indicated that body fat percentage was significantly improved after an eight-week HIFT, while body mass was unaltered . Likewisc review  that indc review ,48,49,50Finally, no significant interaction effect was revealed for any body composition variables. This suggests that HIIT-R and HIIT-F were equally effective in the modulation of body fat percentage.2max was assessed in the present study to estimate the effects of HIIT-R and HIIT-F protocols on aerobic fitness. Running-based HIIT has been shown to increase aerobic capacity in numerous previous investigations. Several studies have reported significant increases in VO2max after HIIT [2max [2max were found after a 6-week HIFT protocol [VOter HIIT ,12,55. Fter HIIT . NeverthIT [2max ,52,57. OIT [2max . Similarprotocol ,59.2max . In line with the magnitude of our results, an increase of 8% in the VO2max was found after a low-volume HIFT [2max observed in the HIFT group was significantly higher than values recorded in previous studies. VO2max has been reported to improve by 5% after a HIFT with no aerobic exercise [2max of 6.3% [2max to HIFT could be explained by the following reasons: firstly, improvements in VO2max were related to the testing modality [2max can be attributed to the fatigue index, which was not measured in our study [In our study, participants from both the HIIT-R and HIIT-F groups experienced improvements in VOume HIFT . It shouexercise . Another of 6.3% . In our modality . Cooper’modality ; secondlmodality ,63. Howemodality ,64. Therur study .2max between the HIIT-R and HIIT-F groups. It is worth noting that running showed higher oxygen consumption for the same intensity compared to other modalities [2max promotion between high-intensity cycling and HIFT. The results from the present study illustrate that functional training is as effective as running for aerobic fitness improvement when performed at high intensity with the same volume and intensity.Although high-intensity running and functional training were both beneficial for aerobic capacity promotion, few studies have compared the effectiveness of these two exercise modalities in terms of aerobic capacity enhancement. In the current study, we controlled for the same intervention intensity and duration and found that surprisingly, there was no significant difference in terms of the changes in VOdalities . Our findalities  that indImportantly, the repetition of sit-ups and the distance of the standing broad jump were significantly increased after HIIT-F, whereas both parameters remained unaltered in the HIIT-R group. Moreover, significant interaction effects were observed in terms of the effects on abdominal and lower limb strength and duration. Our finding is consistent with other HIFT studies. A significant increase in muscle performance after 6 weeks of HIFT was reported, whereas no increase was found in HIIT group using rowing as the exercise modality . SignifiIt is worth noting that the number of flexed push-ups that was completed in the repetition exercise was unchanged in both groups. The unchanged results are in contrast with findings from other investigations. Findings from recent studies revealed increased upper body strength and endurance after functional training executed at a high intensity ,26,27,28The effects of HIFT on muscle performance varies across exercise design and test methods. HIIT significantly increases the proportion of type I fibers , while mA general limitation in the HIFT investigation was the different types of functional exercises that were included. The results might be dissimilar if HIFT was performed with other combinations of movements. Furthermore, the results of our study came from a small sample size and a non-exercising control group was not used. Finally, dietary intake was not controlled during the intervention, and the total calories consumed were not calculated. In addition, the fatigue index was not measured during the aerobic test.Twelve weeks of high-intensity training based on running or functional exercises were both effective in reducing body fat percentage and improving aerobic capacity among healthy inactive females. Relative to running-based high-intensity training, HIFT shows an equally effective alternative with more exercise enjoyment and much stronger adherence regarding body composition and aerobic fitness promotion. Additionally, HIFT resulted in greater muscle performance increases than running-based high-intensity training, after which no gains were observed in terms of muscle fitness.HIFT with self-selected intensity represents an alternative to high-intensity interval running for eliminating exercise barriers for physical exertion. Furthermore, HIFT can be performed anywhere at any time, which limits the barriers of lacking time/money. Finally, HIFT reveals strong exercise adherence and more enjoyment among females. It may be helpful for individuals to promote physical activity and the associated benefits of a prolonged healthy lifestyle.


Background and Purpose: Edoxaban exposure varies across different ethnicities. The purpose of our study was to examine the risk factors associated with high or low edoxaban concentrations in Asian populations.Methods: Participants with atrial fibrillation who were undergoing edoxaban therapy were enrolled. Peak (1–4 h after edoxaban administration) and trough (24 ± 4 h from the last edoxaban dose) blood samples were collected to measure edoxaban concentrations using ultrahigh-performance liquid chromatography with tandem mass spectrometry. The edoxaban concentrations were compared to those observed in clinical trials to define a higher- or lower-than-expected range. Multivariate logistic regression was used to analyze the risk factors associated with high or low edoxaban concentrations.Results: Eighty participants  were enrolled and provided 78 trough and 76 peak samples. Twenty participants (25.6%) were determined to have low trough concentrations, which was associated with higher creatinine clearance and the use of the 30 mg regimen . In contrast, 21 participants (27.6%) had high peak concentrations, which was associated with an off-label overdosing regimen .Conclusion: Our study identified factors associated with increased or decreased edoxaban exposure. The measurement of edoxaban concentration may be recommended for patients with selected characteristics. Edoxaban is a direct, reversible inhibitor of factor Xa that is administered once daily . It dispIn the subanalysis of the Effective Anticoagulation with Factor Xa Next Generation in Atrial Fibrillation-Thrombolysis in Myocardial Infarction 48 (ENGAGE AF-TIMI 48) trial, the edoxaban concentration was consistently lower in Asians than in non-Asians, regardless of whether the edoxaban dose was adjusted . More imThis prospective observational study was conducted at the National Taiwan University Hospital (NTUH) from December 01, 2018, to March 31, 2020. The study protocol was approved by the International Ethics Committee of the NTUH. Each participant provided written informed consent before enrolling in this study. Participants over 20 years of age who had AF and were treated with edoxaban therapy for more than 7 days met the inclusion criteria. Patients who were pregnant or breastfeeding, who failed to provide at least one blood sample, or who declined to provide written informed consent were excluded from this study.®). Participants were asked to refrain from taking the scheduled morning dose of edoxaban before the collection of the trough sample, which was 24 ± 4 h from the last edoxaban dose. The edoxaban administration time on the day before the study date was self-reported by the participants and was used to determine the time that had elapsed from the last edoxaban dose. After the trough sample was collected, participants were asked to take an edoxaban dose under the supervision of a pharmacist and then stay in the hospital until peak sample collection, which occurred 1–4 h after edoxaban administration.Two blood samples were collected through venous puncture and stored in tubes containing K2EDTA  mode. Two transitions were selected as a quantifier and qualifier: 548.1→ 366.1 and 548.1 → 152 for edoxaban and 554.1 → 372.1 and 554.1 → 158.1 for [d6]-edoxaban, respectively.Briefly, an Agilent 1290 UHPLC system coupled with a mass spectrometer  and a Kinetex reverse-phase core-shell C18 column  were used for the concentration analysis. The mobile phase consisted of 0.1% formic acid and 10 mM ammonium acetate in water for solvent A and 0.1% formic acid and 10 mM ammonium acetate in isopropanol and ACN  for solvent B. The flow rate was 0.35 ml min2-VASc score, : demographic characteristics (age and sex), (2), comorbidity conditions , (3), order details and concurrent medications , and (4) laboratory data . The risk of thromboembolism was estimated using the CHADSc score, , and thec score, . The iteBased on the ELDERCARE-AF trial, we also classified all the participants into fragile and nonfragile groups and compared the differences in edoxaban concentrations . ParticiEdoxaban adherence was measured using a self-report questionnaire. An investigator recorded whether the edoxaban dose was skipped for 7 days before blood concentration monitoring. The frequency of a missed dose of edoxaban during treatment was also recorded, along with the reason the dose was skipped. Good adherence was defined as no missed dose 1 week before blood sample collection and seldom skipped edoxaban dose during treatment (≤1 time per month). We then analyzed the effect of edoxaban adherence on drug concentrations.Participants were stratified into on-label dosing, off-label underdosing and off-label overdosing groups to compare the differences in edoxaban concentrations. The definitions for on- or off-label dosing regimens were determined according to the package insert . The staThe study endpoint was factors influencing lower-than-expected-range trough concentration and higher-than-expected-range peak concentrations. We focused specifically on out-of-expected-range concentration because the associations between low concentration and increased risk of ischemic stroke and high concentration and increased risk of major bleeding have been reported in clinical trials . To defiA descriptive analysis was performed to obtain the means, standard deviations, medians, interquartile ranges (IQRs), numbers, and proportions. One-way ANOVA, Kruskal-Wallis H-test or Chi square test was used to compare between groups differences, as appropriate. Post hoc test for pairwise comparison was performed with Scheffe Test to determine which groups were different. Multivariate logistic regression was used to investigate factors associated with lower- or higher-than-expected range edoxaban concentration. In addition to factors which displayed significant difference between groups, we also adjusted age, sex, weight, CrCL and edoxaban dose/appropriateness of edoxaban dose in the model. According to the rule of thumb, every 10 participants is required for adding one covariate to the model in general. Therefore, we determined five pre-specified factors having impact on edoxaban concentration in the multivariate logistic regression analysis should be reasonable. Data were analyzed using IBM SPSS Statistics . The level of statistical significance was set to 0.05.2-VASc and HAS-BLED scores were 3.9 ± 1.6 and 2.3 ± 1.1 points, respectively. Among all the participants, 43 (53.8%) were prescribed 30 mg daily. The baseline characteristics for all participants were listed in The participant enrollment process is shown in The average trough edoxaban concentration was 24.9 ± 21.9 ng/ml measured from 24.9 ± 1.4 h after the last edoxaban dose. Twenty participants (25.6%) were identified to have lower-than-expected-range trough concentrations and nine participants (11.5%) were determined to have higher-than-expected range trough concentration. The comparisons of 3 trough groups were listed in The average peak edoxaban concentration was 239.7 ± 111.0 ng/ml, and was measured at 2.0 ± 0.2 h after edoxaban administration. Twenty-one participants (27.6%) had higher-than-expected-range peak concentrations and 6 participants (7.9%) had lower-than-expected peak concentrations. The comparisons of 3 peak groups were listed in p = 0.90). The proportion of participants with poor adherence with lower-, within-, or higher-than-expected range peak concentration was not significantly different. And the average peak concentration was similar between poor and good adherence groups .Twelve participants (15.6%) had poor edoxaban adherence, included 9 participants (18.8%) had within range trough concentration, 2 participants (10.5%) had lower- and 1 participant (11.1%) had higher-than-expected-range trough concentration. The difference was not significant, as listed in p = 0.01) and were more likely to have higher-than-expected-range peak concentrations  than those with on-label dosing regimen. Nevertheless, the trough concentration was not significantly different between the three groups. The comparison between participants prescribed the off-label and on-label dosing regimens is listed in p = 0.01 for CrCL and 5.77 , p = 0.02 for 30 mg regimen). To predict higher-than-expected-range peak concentrations, the impact of using an off-label overdose regimen was significant .The results of multivariate logistic regression of out-of-expected-range edoxaban concentration are listed in p = 0.01).In addition, we also examined the characteristics of participants with higher-than-expected-range trough concentrations and investigated factors associated with high trough levels, as the information was clinically important to reflect accumulation. To predict higher-than-expected-range edoxaban trough concentration, body mass index (BMI) was the only significant factor , but the difference was not significant (p = 0.23). Similarly, the difference for peak concentration between fragile and non-fragile participants was not significant . None of the participants in fragile group had lower-than-expected range trough or peak concentrations, in contrast to 28.2% of trough concentrations and 8.7% of peak concentrations in fragile group were lower than the expected range. But the differences were non-significant.We further stratified all the participants into the fragile and nonfragile groups according to the criteria used in the ELDERCARE-AF trial. The comparisons between the two groups are listed in This study reported real-world data on edoxaban concentrations in patients with AF. Our data showed that approximately one-fourth of the trough edoxaban concentrations were lower than the expected range reported in clinical trials, and approximately one-fourth of the peak edoxaban concentrations were higher than the expected range. A low trough concentration was associated with good renal function and the use of the 30 mg regimen, while a high peak concentration was associated with an off-label overdosing regimen.2DS2-VASc scores compared to the patients in those two cohorts, the reported edoxaban concentrations were not significantly different between cohorts   cohort in Japan, which enrolled AF patients . Althoug] ng/ml) . HoweverOur data showed that the edoxaban concentration was influenced by the dose regimen. Use of the 30 mg regimen was associated with lower-than-expected-range trough concentrations. To further address whether the edoxaban regimen was appropriate, the results showed that compared to participants prescribed an on-label dosing regimen, those prescribed an off-label overdosing regimens had significantly increased peak concentrations. The dose adjustment criteria for edoxaban are considered to be complicated. Among the participants prescribed off-label dosing regimens, approximately 60% received higher than the recommended dose. More than half of the patients who received the overdose regimen received this regimen due to nonadherence to the CrCL criteria for dose reduction. The association between the appropriateness of the NOAC regimen and clinical outcomes was investigated in a cohort study in Taiwan. The off-label overdose regimen increased the risk of major bleeding compared to the on-label regimen . Our dat2 (2) and 40% of the participants had overweight BMI (25–30 kg/m2), in comparison to 21.4% of patients with normal BMI and 37.6% patients with overweight BMI in the ENGAGE-TIMI 48 trial  to very elderly AF patients with fragile characteristics still reduced the risk of stroke without increasing the risk of bleeding compared with the administration of no treatment . HoweverThe association between the edoxaban concentration and clinical outcome was also reported in the ENGAGE-TIMI 48 subanalysis. In Asian populations with edoxaban trough concentrations less than 15 ng/ml, the risk of ischemic stroke exceeds that of major bleeding. Moreover, when edoxaban trough concentrations are greater than 90 ng/ml, the risk of ICH exceeds that of ischemic stroke . If we aWe acknowledge several limitations of our study. First, this observational study was conducted in a single center, and our results may not be extrapolated to other health care facilities due to differences in patient characteristics. Second, we did not provide pharmacokinetic parameters to support our observation. Nevertheless, only two blood samples were collected during the dosing interval, and thus, the pharmacokinetic parameters of our data may not be accurate. In addition, the dosing regimen for edoxaban is relatively fixed, and the use of pharmacokinetic parameters to precisely determine the dose may not considerably change clinical practice. Third, this is an observational study, and the time of peak and trough sampling varied within a range, which may cause changes in blood concentrations. Last, the sample size was small, and we did not incorporate the outcome analysis. Larger-scale studies with a longer follow-up duration are required to determine whether these observations were linked to worse outcomes.In conclusion, this study provides the first real-world data on the edoxaban concentration in patients with AF. A low edoxaban concentration was associated with better renal function and the use of a 30 mg regimen, while a high edoxaban concentration was related to an off-label overdosing regimen. Physicians should be familiar with the criteria for adjusting the dose of edoxaban and avoid off-label dosing.


To develop and validate a radiomics nomogram based on T2-weighted imaging (T2WI) and apparent diffusion coefficient (ADC) features for the preoperative prediction of lymph node (LN) metastasis in rectal cancer patients.t-test was used for prefiltering. The least absolute shrinkage selection operator method was used for feature selection. Three radiomics scores (rad-scores)  were calculated using the support vector machine classifier. Multivariable logistic regression analysis was then used to construct a radiomics nomogram combining rad-score 3 and independent risk factors. The performances of three rad-scores and the nomogram were evaluated using the area under the receiver operating characteristic curve (AUC). Decision curve analysis (DCA) was used to assess the clinical usefulness of the radiomics nomogram.One hundred and sixty-two patients with rectal cancer confirmed by pathology were retrospectively analyzed, who underwent T2WI and DWI sequences. The data sets were divided into training (n = 97) and validation (n = 65) cohorts. For each case, a total of 2,752 radiomic features were extracted from T2WI, and ADC images derived from diffusion-weighted imaging. A two-sample The AUCs of the rad-score 1 and rad-score 2 were 0.805, 0.749 and 0.828, 0.770 in the training and validation cohorts, respectively. The rad-score 3 achieved an AUC of 0.879 in the training cohort and an AUC of 0.822 in the validation cohort. The radiomics nomogram, incorporating the rad-score 3, age, and LN size, showed good discrimination with the AUC of 0.937 for the training cohort and 0.884 for the validation cohort. DCA confirmed that the radiomics nomogram had clinical utility.The radiomics nomogram, incorporating rad-score based on features from the T2WI and ADC images, and clinical factors, has favorable predictive performance for preoperative prediction of LN metastasis in patients with rectal cancer. More than 700,000 people worldwide were newly diagnosed with rectal cancer in 2018 . AccordiRadiomics based on advanced pattern recognition tools has been considered useful to extract a large number of quantitative features from medical images –16. It cThus, in the current study, we first sought to construct a radiomics score (rad-score) based on features from volume lesion of T2WI and ADC images to distinguish between LN-positive and -negative rectal cancer patients and analyze the discriminative abilities of each imaging model. Then we sought to develop and validate a radiomics nomogram that would incorporate a rad-score based on the combination of T2WI and ADC features, and clinical risk factors to facilitate noninvasive estimation of LN status.The study was approved by the Ethics Review Board of Shengjing Hospital of China Medical University (2020PS011K), and written informed consent was obtained from each patient. In the present study, 236 patients with pathological confirmation of LN status were preliminarily enrolled between September 2018 and August 2020. Seventy-four patients were excluded for the following reasons: (1) patients underwent any treatment before MRI scanning, such as neoadjuvant chemoradiotherapy, endoscopic biopsy, surgery, and so on; (2) image quality was poor due to apparent motion artifacts on the DWI and T2WI sequences. Finally, 162 eligible patients were selected for subsequent analyses. The patients were randomly divided into the training (n = 97) and validation (n = 65) cohorts.The histopathological evaluation of regional LN malignancy was regarded as the gold-standard for LN metastasis. Pathological reports of surgically resected specimens were retrospectively collected from our PACS. The LN was defined as positive when the number of regional LN metastasis was greater than or equal to one, while the absence of regional LN metastasis was recognized as negative.2; slices, 20; slice thickness, 5 mm; spacing between slices, 0.5 mm; and NSA, 2. The parameters for DWI included: TR/TE, 6000/76 ms; flip angle, 90°; matrix size, 288 × 288; FOV, 450 × 450 mm2; slices, 48; slice thickness, 5 mm; spacing between slices, 1 mm; and b values, 0 and 1,000 s/mm2.All MRI examinations were performed in the supine position on a 3.0-Tesla (T) scanner  with an eight-channel phased-array surface coil. There was no bowel preparation or intravenous antispasmodic agents administered. High-resolution rectal MRI protocols included transverse DWI and T2WI, and sagittal fat-suppression T2WI. The acquisition parameters for transverse T2WI included: repetition time (TR)/echo time (TE), 2200/65 ms; flip angle, 90°; matrix size, 288 × 288; field of view (FOV), 250 × 250 mm0-lnSI)/(b-b0), where SI0 and SI represent signal intensity at b values of 0 and 1,000 s/mm2, respectively.DWI and T2WI images were exported from the Picture Archiving and Communication System at our institution. ADC maps were generated using MATLAB 2018a  according to loaded DWI images using the following formula: ADC = (lnSIhttp://www.itksnap.org) (2) first with reference to T2WI, and then automatically turned into the VOI which was copied to the corresponding ADC maps finally , including the whole tumor and excluding obvious necrosis, hemorrhage, gas, and lumen content areas, was independently segmented on T2WI and DWI data by a radiologist , who was blinded to the clinical and pathological outcomes. All VOIs were delineated with an open-source software, ITK-SNAP version 3.8.0 from UPenn (nap.org) . For T2W finally . An overPyRadiomics, an open-source python package for enabling the standardization of image processes and extracting a large panel of radiomic features from medical images, was used to extract radiomic features from T2WI and ADC data within manually segmented VOIs . To avoiWe randomly chose 30 cases of MRI images (T2WI and ADC); VOI segmentation was performed by two radiologists . Feature extraction was performed on the two sets of VOIs generated by the two radiologists to obtain two groups of the radiomic features. Intraclass correlation coefficients (ICCs) were determined using the two sets of radiomic features to evaluate the reproducibility and stability of each feature. We interpreted a coefficient of 0.81 to 1.00 as an almost perfect agreement, 0.61 to 0.80 as a substantial agreement, 0.41 to 0.60 as a moderate agreement, 0.21 to 0.40 as a fair agreement, and 0 to 0.20 as a poor or no agreement .t-test) between LN-positive and -negative groups in the training cohort were selected (https://www.R-project.org).To reduce the feature dimension and remove irrelevant features, two steps were applied for feature selection. First, some features based on univariate statistical tests . Calibration and DCA curves were generated using R software.Univariate logistic regression analysis was first conducted with the following clinical information: age, sex, LN size (maximum LN short diameter), tumor size, tumor location, T stage, and rad-score 3 to identify potential predictors . Then mut-tests for continuous variables, as appropriate. All statistical tests were two-tailed and were conducted with a statistical significance level of 0.05. Statistical analyses were performed and figure plots were generated with R software and SPSS software . The DeLong test was used to statistically compare the AUCs between the models.Univariate analysis was used to compare the differences in clinical and pathological characteristics between LN-positive and -negative groups using the chi-square test for categorical variables, and two-sample P = 0.819).Patient characteristics were summarized in Of the 2,752 radiomic features extracted from T2WI and ADC images, 2,076 were demonstrated to have high stability, with ICCs from 0.8003 to 0.9973.t-tests) as a feature filter in the training cohort. We included more features than those that showed significant differences between LN-positive and -negative groups as compensation to avoid eliminating highly discriminative features in multivariate analyses, rather than univariate analysis (t-tests (P < 0.1) allowed for the selection of 530 features, including 313 T2WI and 217 ADC features. Next, 313 T2WI and 217 ADC features were respectively reduced to seven and 11 potential predictors by applying LASSO logistic regression using 10-fold cross-validation via the minimum criteria. Finally, the combination of the seven T2WI and 11 ADC features was reduced to 13 potential predictors by applying LASSO logistic regression using 10-fold cross-validation via the minimum criteria. Three rad-scores were calculated. The resultant coefficients of features in each group used in calculating the corresponding rad-score were shown in To reduce the number of weak features, we first performed univariate analysis ; the same result was achieved in the validation cohort (P < 0.01), as shown in There were significant differences in rad-score 1, rad-score 2, and rad-score 3 between LN-positive and -negative patients in the training cohort  and validation cohort (P = 0.244). The DCA result for the nomogram was shown in The ROC curves were plotted for radiomics nomogram from the training and validation cohorts Figure 5LN status is the key factor in determining whether to conduct adjuvant therapy or additional surgical resection , 7, 8. T2), which achieved good diagnostic performance in the validation cohort (2)  . TherefoRad-score 1, on the basis of T2WI, was mainly constructed by wavelet features (6/7); this demonstrated that wavelet features better reflected tumor biology and heterogeneity. All wavelet features were derived from the decompositions and the approximation by wavelet filter to the original image. Image transformation using a filter can eliminate noise or sharpen the image and does not change the semantics of the features . Therefot-tests). We found that associating a single radiomic feature with complex tumor biological processes remained a challenge. Therefore, it was more common to combine the panels of selected features into a rad-score. Our results showed that the developed rad-score 2 could achieve good performance and yielded an AUC of 0.770 in the validation cohort. A recent study on breast cancer reported the potential values of higher-order statistics features in predicting sentinel LN metastasis  and six ADC  features, and indicated that radiomic features on T2WI and ADC maps had good performance in predicting LN status. According to the AUCs, the rad-score 3 obtained the highest score among three rad-scores in predicting the LN status. Recently, several studies also reported that radiomics models based on multiparametric MRI data could improve the predictive performance for tumor characteristics , 42, 43.In multivariate logistic analysis, the rad-score 3, age, and LN size were identified as independent parameters of LN metastasis. We found that the LN positive group had a significantly younger age compared with the LN negative group which was consistent with that of the study conducted by Li et al. . This reA recent study showed that MRI radiomics based on multi-regions  could improve efficacy in the identification of LN metastasis in patients with rectal cancer . HoweverThere were several limitations to this study. First, a bias of selection might exist because the study used a retrospective design. Second, the patient sample size was small and all cases were derived from a single institute. Multi-center studies with a larger sample set are required to further validate our model. Third, the segmentation of 3-D lesions was performed manually, which was time-consuming and complicated for the larger sample sizes. Thus, a fully automatic analysis method for rectal lesions with favorable reliability and reproducibility should be developed in further studies.In conclusion, our study demonstrated that the radiomics nomogram, incorporating rad-score based on features from the T2WI and ADC images, and clinical factors, has potential for the preoperative identification of LN status. Although the results were satisfactory, the model should be validated by further studies with larger sample sizes from multiple centers to evaluate the performance.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Shengjing Hospital of China Medical University. The ethics committee waived the requirement of written informed consent for participation. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CL conducted data analysis and manuscript writing. JY was responsible for the manuscript revision. All authors contributed to the article and approved the submitted version.This work was supported by the Research and development (R&D) foundation for major Science and Technology from Shenyang (No. 19-112-4-105), Big data foundation for health care from China Medical University (No. HMB201902105), Natural Fund Guidance Plan from Liaoning (No. 2019-ZD-0743), and 345 Talent Project from Shengjing Hospital of China Medical University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sections were stained with hematoxylin and eosin and Masson trichrome stains. Immunohistochemistry labelling was used to indicate scars (α-smooth muscle actin [α-SMA] and collagen-1) and angiogenesis (von Willebrand factor). Also, the macrophages inflammatory protein-3α (MIP-3α) indicates an early inflammatory process. The post dressing of the AB group demonstrated hair follicle remains and adipose tissue development. The NHI group appeared with a denatured matrix. Complete healing was seen in the AB group after 28 days with skin appendages similar to normal, while the NHI group showed no appendages in the centre of the actively inflamed area. The α-SMA was found in both groups. Collagen-1 was highly expressed in the NHI group, which led to a scar. Angiogenesis was found more in the AB group. The AB group had shown the capacity to accelerate complete healing and recover skin appendages better than the current protocol.Burn is a common case in developing countries, with over half of fire-related deaths reported in Southeast Asia and full-thickness burns as a high mortality risk. Human amnion has been used as a wound dressing for centuries. In this study, a decellularised amnion overlaid with fibrin, “amnion bilayer (AB),” was used as a dressing immediately after burn and as a graft to replace the scar in Sprague-Dawley rats subjected to full-thickness burn model. The aim was to observe whether amnion bilayer can reduce damages in third-grade burn when skin replacement is deemed impossible. The burn was induced using an electrical solder, heated for 5 mins, and contacted on the rat’s bare skin for 20 s. AB was applied as a (i) dressing immediately after induction and graft after eschar removal. Two groups (n = 6) were compared: AB and Sofra-Tulle  Burns are primarily found in developing countries, and half of the incidents were reported in Southeast Asia . The WorAccording to the depth of destruction, there are three grades of burn: superficial (grade 1), partial (grade 2A) or deep partial-thickness (grade 2B), and full-thickness (grade 3) burns . Grade 1In general, wound healing involves several stages: inflammation, granulation tissue formation, re-epithelialisation, and remodelling. This complex process is regulated by the complex signalling molecule involving numerous growth factors, cytokines and chemokines . CytokinA full-thickness burn needs surgical debridement to remove the eschars to prevent infection and optimise treatment . PatientIn this study, the amnion bilayer (AB) was applied as a dressing immediately after burn induction, followed by AB as graft replacement started from Day 7 after scar removal. AB is a decellularised human amnion membrane (hAM) overlaid with fibrin. The hAM naturally has anti-inflammatory and antibacterial capacities . The hAMThis study aimed to investigate the application of AB as a dressing for full-thickness burns in rat models. In a scenario where health care facilities are insufficient, and an eschar removal is yet possible, a dressing is needed to allow transportation to the higher facility. AB was investigated for its capacity to reduce the damages when applied immediately after severe burn until skin transplantation is available.According to the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. The ethical board of the Faculty of Medicine, University of Indonesia (UI), had approved all procedures on this study . The methods to induce burn wounds were done following a previous study [ad libitum. Three groups were compared. Group 1 was for the no treatment, Group 2 was treated with AB, and Group 3 to the standard protocol applied in the National Hospital of Indonesia (NHI); Sofra-Tulle®, as a dressing to full-thickness burn patients when skin grafts are impossible. All interventions such as burn induction, graft dressing and transplantation, or termination were performed under general anaesthesia. The burn induction method was done following a previous study [Rat as burn model has been well established in the literature . Eighteeus study .On the first day of the intervention, rats were weighed, then anaesthetised with intramuscular injection of a combination of 100 mg/kg body weight ketamine and 10 mg/kg xylazine  . After 5The Methods of euthanasia followed the AVMA Guidelines on Euthanasia, ACLAM Task Force Guidelines on Euthanasia . ChemicaThe hAM was obtained from caesarean section procedures. The donor was nonreactive to human immunodeficiency virus (HIV) and hepatitis B and C. The hAM then separated from the chorion, washed consecutively using Triton-X  and sodium dodecyl sulphate , coated by fibrin, and then incubated at 37°C for 24 h. All grafts were randomly checked for microbiological tests (sterility assays). The test used tryptone soy broth , Sabouraud dextrose agar , and blood agar  to verify the biocompatibility towards humans bone-marrow MSC and acellularity using H&E  and DAPI staining . All grafts were found acellular, had no growth, and were biocompatible.2) for 20 seconds. The electrical solder was initially heated for 5 minutes. The eschar size was measured, and then AB was applied as a dressing (n = 6) for seven days, covered with gauze, and suture to keep the AB in a place. The NHI group was treated similarly to the AB group, however Sofra-Tulle ® was used as the dressing. The eschar was removed on Day 7 in both groups, replaced with AB or Sofra-Tulle ®, respectively. The termination was conducted on all rats once a rat had been shown to recover fully. Throughout the study, records of rats’ behaviour, weight, and wound size were observed every day. The wound-size reduction was calculated using the formula below, as published by Chen et al., 2008 [Once confirmed anaesthetised, the rats were induced for burn wound, followed by the previous study , 26. Bril., 2008 :AreaofFor histology, all specimens were fixed in 10% (v/v) neutral-buffered formalin solution  for 48 h, processed automatically in a tissue processor , paraffin blocked, and sectioned by 5 μm thickness. Sections were stained using hematoxylin and eosin  and Masson trichrome stains  according to the manufacturer’s instructions, visualised using the brightfield microscope, and captured using Zeiss Axio Imager .All immunohistochemical labelling followed the biotin-avidin labelling protocol against several markers. The collagen-1  and α-smooth muscle actin  indicated the scar formation. The Von Willebrand factor  indicates angiogenesis (22). The macrophages antigen  show an early inflammatory process. Antigen retrieval was performed using heat-induced epitope retrieval with 10 mM citric acid pH 6 , Tris-EDTA pH 9  and the enzymatic reaction using proteinase K  at 25°C for 10 min (collagen-1 and vWF).3 and stored in RNAlater solution  until all samples were collected. The RNA was isolated using TRIzol™ Reagent  and converted into complementary DNA using SensiFAST™ cDNA Synthesis Kit . The gene expressions were identified using the real-time qPCR method using SensiFAST™ SYBR Lo-ROX Kit  and performed with the AppliedBioscience-7500 Fast Dx Real System PCR instruments . The cytokines to indicate inflammation, such as TNFα, IL-6, MMP-1, and anti-inflammatory IL-10, were identified. The primer sequence used in this study are IL-6 , IL-10 , MMP-1 , TNF-α , and GADPH as the reference gene . All PCR data were calculated using Livak analysis.All specimens were cut into 5x5x5 mmimageJ.NHI.gov). The method to analyse wound area was done following a previous study [The images of the burn wound were documented daily using a digital camera , captured from a consistent height and distance to the object, and a ruler was placed next to the wound and used as a reference pixel scaling while processing the image in ImageJ software  and Day 14  showed that the pro-inflammatory genes of the NHI group were highly expressed very significantly compared to the AB group, also increased significantly from Day 0 .A rat skin has a substantial skin layer similar to humans; however, the mechanism of wounds healing in rats by contraction, while humans through epithelialisation that heals slower , 31. Eve®, were observed to apply as a dressing.An eschar was formed after burn induction immediately. A study reported that wound healing up to three weeks increased scar formation up to 33% . TherefoOur study showed that the application of AB immediately after burn for seven days improved the damage severity due to heat and promoted the restoration of skin appendages, compared to the standard protocol. The rats in the AB group appeared to be calm. After the treatment, able to maintain normal activities such as eating, urinating, and grooming. Reports indicated that pain causes animal labs to be aggressive , 37. Ini®). The contraction rate in the wound healing process in rats is influenced by several factors, including age, treatment and depth of burn wound.In contrast, the AB group rats were calm and responded similarly throughout the experiment while weighing. Although both groups did not differ significantly, except the weight of rats in the AB group after Day 7 until 14 was slightly higher, we assumed AB application might reduce pain compared to the NHI group. The wound healed significantly faster in the AB group by days 10 and 14. These happened once the scar was removed and replaced by a graft  is the most common pathogen observed in burn patients, unresponsive to antibiotic treatments as it forms biofilms on wounds. Nonetheless, an acute stage of this infection results in an overactive immune response that induces a significant inflammation response [After seven days of burn induction and dressing, the NHI group’s macrophage inflammatory protein-3 α (MIP-3-α) was strongly expressed. A superficial layer fully populated with mononuclear cells contrasts with the AB group. MIP-3 α cells are recruited, especially to an infected area and also found in a prolonged inflammatory response. These can disrupt the normal progression of wound healing . Accordiruginosa . P. aeruresponse –46. In oStreptococcus group A, Pseudomonas aeruginosa [Our study was terminated once the rats from any group showed to heal completely. After 28 days, no infection was observed in the AB group, while the wound in the NHI group remained active at the same time point. The infection was not observed until Day 28 . The AB ruginosa . Antimicruginosa . The decruginosa . The cenThere are several markers to identify wound healing and scar formation. Immunohistochemistry labelling against collagen-1 indicates mature fibre. The α-SMA marks myofibroblastic in the early scar process but also the smooth muscles around blood vessels. Sections with α-SMA and vWF positive indicate angiogenesis and functional endothelial. During wound healing, fibroblasts will transform into myofibroblasts, as indicated by α-SMA. Myofibroblasts have a strong contractile force to retract the wound size . The recIn our study, the α-SMA expression was distributed evenly in the dermis, while the NHI group had less. Perhaps, the implantation of AB graft also recruited mesenchymal stromal cells to the injured site and induced angiogenesis. Angiogenesis is indicated by α-SMA expression identified from the smooth muscle in the wall of the newly formed blood vessels and the vWF labelling the endothelial. Our study showed that both α-SMA and vWF were expressed in the AB group, whilst the NHI group was also found. Report on decellularised hAM, overlaid with electrospun nanofibrous silk fibroin, stimulated neovascularisation and re-epithelialisation . MoreoveNext, the epidermis of the NHI group had been thickened, compared to the normal skin or the AB group, while the collagen deposition was irregular. These might indicate a prolonged inflammatory response .Pro-inflammatory cytokines such as IL-6 and TNF- α were reported to be increased during the early wound to initiate the healing response , usuallyIn this study, the gene expressions of the pro and anti-inflammatory markers of NHI and AB groups wounds. The pro-inflammatory gene expressions increased significantly in the NHI group; TNF-α by 7-fold, IL-6 by 5.5-fold, and MMP-1 was up to 9-fold . The repOthers showed that burn had a significant increase of IL-1α, GM-CSF, TNF, IL-6, IL-17, G- CSF, KC, MIP-1α, and RANTES expressions. The anti-inflammatory cytokines  were initially decreased within the first 9 hours, then increased after 24 hours, followed by the normal range by day 10. The burn and sham mice showed no difference in expressing IL-1-α, IL-12p40, whilst the IL-4 was undetectable . This reSeok et al. (2013) analysed the relationship between gene expressions between humans and rats, focusing on a burning study. Pearson correlations (R2) of 40 most dominant pathways and suppressing after burn-in rats and humans; indicated that the percentage of genes and median correlations developed to the same direction were 67% and 0.16, respectively. The gene response time in both humans and rodents started by the first 6–12 h; however, the healing duration differed enormously. The rodents’ genes were stabilised by the first four days; in contrast, humans needed at least six months . On the The AB group showed complete healing after 28 days of experiment or 21 days post-transplantation. The fibrin might contribute to this healing capacity in the amnion bilayer, generated from platelets rich plasma (PRP). PRP contained rich in growth factors such as fibronectin, vascular endothelial growth factor (VEGF), insulin growth factor (IGF-I) and interleukin -1b (IL-1b) . Others Wound healing processes are a cascade of inflammation, proliferation, and re-epithelialisation/remodelling , 51. InfThe healing rate of a full-thickness burn in neonatal rats was reported faster than the adulthood, as the neonatal rats took three days, while adults up to 30 days or longer . The re-Our study showed that statistical analysis of wound size was different significantly between NHI and AB groups. The report indicated that EGF  promoted re-epithelialisation and wound closure processes . Some adIn this study, a full-thickness burn was successfully modelled in rats and biological matrix application. In conditions when skin transplantation is delayed, AB as a dressing showed to be encouraging. AB application as a graft to replace the skin also shown to heal faster than the standard protocol when skin transplantation is not available with full recovery after 28 days, without a scar, and with skin appendages developed. The epidermis was slightly thicker than the normal skin but not as thick as the group without graft.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


The good compatibility of the unloaded and CBD-loaded PMs is assessed in a human corneal epithelial cell line. Then, we confirm the permeability of CBD-free PMs and nanoencapsulated CBD in human corneal epithelial cell monolayers under liquid–liquid and air–liquid conditions. Overall, our results highlight the potential of these polymeric nanocarriers for ocular drug delivery.Ocular drug delivery is challenging due to the very short drug residence time and low permeability. In this work, we produce and characterize mucoadhesive mixed polymeric micelles (PMs) made of chitosan (CS) and poly backbones graft-hydrophobized with short poly(methyl methacrylate) blocks and use them to encapsulate cannabidiol (CBD), an anti-inflammatory cannabinoid. CBD-loaded mixed PMs are physically stabilized by ionotropic crosslinking of the CS domains with sodium tripolyphoshate and spray-drying. These mixed PMs display CBD loading capacity of 20%  Cannabis sativa has emerged as a promising therapeutic agent to treat a broad spectrum of local and systemic diseases  (ATCCw/v dispersion prepared in water supplemented with acetic acid of pH 5.5) were diluted in transport medium  buffered to pH 7.4 with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid  and NaHCO3  to the final concentration (0.01% and 0.03% w/v). For this, mixed PMs were prepared with a CS-PMMA30:FITC-labeled PVA-PMMA16:PVA-PMMA16 weight ratio of 1:0.4:0.6. For non-covalent crosslinking, a 1% w/v TPP solution in HBSS was prepared and 3 µL of TPP solution per 1 mL of PM dispersion was added at least 6 h before the permeability experiment. Fluorescently labeled mixed PMs that were spray-dried and redispersed in HBSS were also used.A permeability assay was conducted with mixed crosslinked PMs in hCEc monolayers, an in vitro model of the human cornea . For thi5 cells/well) on cell culture inserts  maintained in 12-well plates  with 0.5 and 1.5 mL of keratinocytes serum-free medium (see above for culture of hCEc), to the apical and basolateral, respectively. The culture medium was replaced every 2–3 days and the integrity of the hCEc monolayer was characterized by transepithelial electrical resistance (TEER) measurements performed with an epithelial volt-ohm-meter EVOM2 .Experiments were performed 14–21 days post-seeding of hCEc  and basolateral (1.5 mL) compartments was replaced with HBSS without PMs and the cells incubated for 20 min at 37 °C. Then, the transport medium in the donor  compartment was replaced by the sample (0.4 mL) containing the PMs and in the acceptor  compartment by fresh transport medium (1.2 mL). Plates were incubated on an orbital shaker  at 37 °C and gently shaken to minimize the impact of the unstirred water layer. After 5, 10, 15, 30, 45, 60, 90, 120, 180, and 240 min, medium (600 μL) was extracted from the acceptor compartment and replaced by the same volume of fresh transport medium to maintain the total volume constant. The extracted medium was used for quantification of the transported PMs by fluorescence spectrophotometry in a Fluoroskan Ascent Plate Reader  using black 96-well flat bottom plates  at wavelengths of 485 nm for excitation and 538 nm for emission according to a calibration curve of non-crosslinked and crosslinked PMs in HBSS in a concentration range between 0.0001% and 0.1% w/v . The apparent permeability (Papp) was calculated according to Equation (2)2). Results are expressed as mean ± S.D. of at least three independent experiments.The permeability assay was conducted in two corneal epithelium models with air–liquid (AL) and liquid–liquid (LL) interface. Eight days after culture, inserts were lifted to produce an AL culture or left in culture medium for a LL one and cultured for 1–2 more weeks. Only inserts where the TEER was >140 Ω·cmAt the end of each experiment (240 min), 50 μL was also removed from the donor compartment to calculate the mass balance by fluorescence spectrophotometry (see above). The percentage of PMs retained by the cell monolayer (%PMs inside cells) was estimated according to Equation (3)w/v CBD-loaded PMs (20% w/w loading) were diluted to a final total copolymer concentration of 0.03% w/v , and the permeability assay was conducted under AL and LL setups. After 5, 10, 30, 60, 90, 120, 180, and 240 min, 600 μL of medium was extracted from the acceptor compartment and replaced by the same volume of fresh transport medium to maintain the total volume in the chamber constant. The extracted medium was used for quantification of the transported CBD-loaded PMs by diluting them with ethanol (2 mL) to extract CBD and conducting the Beam test for which a CBD calibration curve in ethanol in a concentration range between 0.0001% and 0.1% w/v (R2 = 0.9992) was built. At the end of the experiment (240 min), 50 μL was also removed from the donor side of each sample to calculate the mass balance, as described above. Results are expressed as mean ± S.D. of at least three independent experiments.We also measured the permeability of CBD-loaded PMs by tracking the gradual increase of the CBD concentration in the acceptor compartment over time by the Beam test (see above). For this, fresh CBD-loaded mixed PMs were prepared as described above in HBSS instead of water without any fluorescence labeling of the PMs. Briefly, 0.1% t-test on raw data . p-values of less than 0.05 were considered as statistically significant.Statistical analysis of the different experiments was performed by N-acetylcysteine  eye drops have been developed as a medical device and clinically trialed for the treatment of dry eye with promising results [Mucoadhesive polymers have been investigated in the development of advanced local and trans-mucosal drug delivery systems . CS is a results . PMs can results . CS emer results . CS slow results .2 [2) [PVA is a synthetic water-soluble polymer broadly used in a wide range of food, biomedical, and pharmaceutical applications with very good biocompatibility . PVA con2 , and gre2 [2) .g-oligo(N-isopropylacrylamide) and CS-g-PMMA PMs in different cell types by crosslinking them ionotropically [g-PMMA [CS is a polycation under physiological conditions, and cell toxicity has been associated with its electrostatic interaction with the cell membrane. However, its cell compatibility can be improved upon partial neutralization of the positive charges by ionotropic crosslinking with polyanionic molecules , by decreasing the degree of deacetylation or by chemically modifying or mixing it with other polymers that reduce the availability of cytotoxic free primary amine groups on the nanoparticle surface ,62. We iopically ,43, and  [g-PMMA . These m [g-PMMA ,63.g-PMMA and PVA-g-PMMA PMs produced by the co-micellization to encapsulate CBD for trans-corneal delivery. The use of PMMA was supported by its broad use as pharmaceutical excipient in oral drug delivery systems [In this conceptual framework, we anticipated the potential of mixed CS- systems ,65 and i systems ,67,68. Sg-PMMA and PVA-g-PMMA copolymers by the free radical graft polymerization of MMA onto the backbone of both CS and PVA, respectively (1H-NMR (We synthesized CS-ectively . Copolym (1H-NMR  and the g and Tm of PVA was detected at 78 and 196 °C (ΔHm = 28.8 J/g), respectively [g at 87 °C and Tm at 179 °C (ΔHm = 24.2 J/g). In both copolymers, the thermal transition of PMMA blocks could not be detected , as exemplified in w/v CBD-free crosslinked mixed PMs. The Dh of the PMs ranged between 96 ± 6 nm for CBD-free non-crosslinked and 151 ± 8 nm for CBD-loaded crosslinked ones  [w/w CBD showed a broad exothermal peak in the 240–270 °C range associated with CS decomposition  , while Cposition . In addim of PVA was not detected in the mixed PMs, indicating that it is amorphous  after spray-drying. Since the crosslinking with TPP is not covalent, we did not expect to observe major differences between non-crosslinked and crosslinked PMs by DSC. The DSC thermogram of mixed PMs was a combination of those of the individual components, though the Tmorphous .w/v) and spray-dried it under the same conditions used for the mixed PMs. Then, spray-dried pure CBD was collected, and Beam test was conducted on three samples. The CBD content in the spray-dried samples was 98.5%, indicating that CBD withstands the spray-drying conditions.Drugs nanoencapsulated within hydrophobic polymeric nanoparticles are usually amorphous owing to the formation of solid solutions. The absence of a CBD melting endotherm in CBD-loaded PMs confirmed that this compound is amorphous . As menth measured by DLS and NTA because the latter methods measure the hydrodynamic size that comprises also hydration water.The spherical morphology of the CBD-free and CBD-loaded mixed PMs was visualized by cryo-TEM . Non-croDrying methods such as freeze-drying have been investigated to improve the long-term physicochemical stability of nanoformulations. In the case of the freeze-drying of nanoparticles , the addition of cryo/lyoprotectants in relatively large relative amounts is often required to preserve their original size upon redispersion . OtherwiFreeze-dried non-crosslinked mixed PMs were rounded and in the nanometer scale range .w/v, and the Dh and PDI of the PMs was measured by DLS. Results are summarized in However, as usual after freeze-drying of nanoparticles made of polysaccharides such as CS, they formed a polymeric network in which PMs are strongly bound to each other and they could not be easily visualized a. No majh did not dramatically change , the PDI values increased sharply, this phenomenon being more noticeable for CBD-loaded mixed PMs (from approximately 0.20 up to 0.73), meaning that these PMs form stable aggregates upon freeze-drying  for 4 and 24 h and the viability estimated by the MTT assay. The lowest PM concentration (0.05% w/v) showed viability values ˃70% except for spray-dried and redispersed CBD-loaded non-crosslinked PMs that exhibited 65 ± 8% after 24 h . Non-crosslinked Pms were not sued because these concentrations are below the CMC of both copolymers [Cell viability values ˃70% comply with the guidelines of ISO 10993-5 for the evaluation of the cytotoxicity of medical devices in vitro. The cell viability loss increased for 0.10% ocessing b. When tied ones c. Viabilidelines a,b. Basepolymers ,44,45.Since the self-assembly of CS-PMMA30 and PVA-PMMA16 to form mixed PMs is random, the use of FITC-labeled PVA-PMMA16 in their preparation ensured the fluorescent labeling of all the PMs and preserved free amine groups in the sidechain of CS for ionotropic crosslinking with TPP and interaction and transient opening of tight junctions in epithelial monolayers.app under LL and AL conditions [w/v) above the CMC, crosslinked and then, diluted to final concentrations of 0.01% and 0.03% w/v, which are below the CMC of these copolymers. Non-crosslinked PMs were not used because they could disassemble upon dilution. To estimate the integrity of hCEc monolayers, TEER values were measured over 14–21 days until a constant value between 140 and 170 Ω·cm2 was measured for LL and AL conditions, respectively. An increase in the TEER value of 25–30 Ω·cm2 under AL incubation conditions on the same experiment day was consistent with the formation of stronger tight junctions by these epithelial cell lines, which might reduce the nanoparticle permeability across it. This comparison is physiologically relevant because the outer cells of the human corneal epithelium are exposed to air and covered by a thin layer of mucus that preserves their hydration state, what usually results in the formation of more and stronger tight junctions [app was greater for LL than for AL interface owing to the formation of stronger tight junctions by the latter model, as supported by TEER measurements  [app between 34 and 60 × 10−7 cm/s) [First, we characterized the permeability of CBD-free mixed PMs in a corneal epithelium model by measuring the Pnditions . For theunctions . This beunctions . As expeurements . This pe0−7 cm/s . The sam pathway . Moreove−7 cm/s)  and the −7 cm/s) . These d−7 cm/s) .At the end of each permeability study, we measured the fluorescence in both the donor and the acceptor chamber to calculate the mass balance and estimate the percentage of CBD-free mixed PMs retained by the cell monolayer due to unspecific adsorption or cell internalization, this percentage being always <12%, in good agreement with results reported elsewhere . Based ow/v CBD-loaded fresh crosslinked mixed PMs containing 20% w/v CBD loading under LL and AL conditions. To evaluate the permeability of CBD encapsulated within mixed PMs, we did not label the PMs with FITC because CBD was extracted from the samples and the concentration measured by the Beam test (see above).After assessing the permeability of CBD-free PMs, we characterized the permeability of 0.03% w/v .Results of these experiments indicated that fresh CBD-loaded crosslinked mixed PMs cross this model of corneal epithelium in vitro. As expected, the permeability rate under LL conditions was faster than under AL ones due to the formation of stronger tight junctions in the latter. For example, at the end of the permeability assay (4 h), 82% and 53% of the encapsulated CBD was measured in the acceptor compartment for LL and AL setups, respectively . These rIt is also important to stress that our model of corneal epithelium is deprived of the outer mucus layer that covers the cornea. Our PMs are mucoadhesive and this feature is expected to prolong their residence time with respect to non-mucoadhesive ones, which in turn, could increase the amount of drug delivered across the cornea over time. As mentioned above, CBD-loaded crosslinked mixed PMs are larger than the unloaded ones though still in the size range that fits trans-mucosal paracellular transport. Overall, our results support the promise of these CBD-loaded nanocarriers as a drug delivery platform to the cornea and the inner eye.w/w was achieved by using a simple and reproducible microfluidic system. Mixed PMs showed monomodal size and narrow size distributions with sizes in the 100–170 nm range and spherical morphology, as visualized by cryo-TEM. In addition, mixed PMs were successfully stabilized physically by non-covalent crosslinking and the size of crosslinked counterparts increase slightly though it remained <200 nm, fitting mucosal drug delivery. Then, the cell compatibility of the different PMs was evaluated by using a human corneal epithelial cell line that serves as a model of outer surface of the eye. CBD-free and CBD-loaded mixed PMs showed good cell compatibility before and after the crosslinking. Additionally, we investigated the ability of the mixed PMs to cross models of epithelium in vitro and measured the Papp of the mixed PMs in confluent hCEc monolayers under LL and AL conditions. Permeability across corneal epithelium is a crucial step to ensure efficient ocular drug delivery. Findings showed that these mixed PMs cross this corneal epithelium model even under AL conditions, at which cells form stronger tight junctions most probably by transiently opening them (paracellular mechanism). Finally, these results were supported by showing the efficient permeability of CBD across both LL and AL models. Overall, our results support the promise of these PMs as a drug delivery platform for the treatment of eye diseases.This work reports on the synthesis and characterization of mucoadhesive mixed CS-PMMA30:PVA-PMMA16 PMs (1:1 weight ratio) for the loading of CBD and trans-corneal delivery of CBD for potential application in inflammatory eye conditions. A high CBD loading of up to 20%